ABSTRACT Vitamins and other dietary supplements have been purported to boost the immune system, prevent cancer, and extend life. However, multiple studies have shown contrasting data for supplement use in cancer prevention, cancer treatment, anti-aging, and well-being. Cancer, infection, neurocognitive degeneration, and other conditions prominent in the aging human population are often characterized by an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species. And with aging, comes a decline in the levels of cofactors and coenzymes that function in these metabolic pathways that moderate and buffer oxidative stress in healthy cells. Taken together with our parent R01 that investigates the dysfunction of NADK in pancreatic cancer (PaCa), we were prompted to carefully consider the commonly used cofactor supplement, NAD+, taken under the premise of improving health and preventing cancer. Our data have shown that PaCa etiology may involve dysregulation of NADK and allied pathways in NAD+ metabolism, resulting in altered metabolism, reducing ROS and increasing the production of NADPH, supporting cancer cell growth. However, these findings are in contrast to the concept of NAD+ supplementation as an ?anti-aging therapy? and in cancer prevention. We purport that cofactor balancing is critical for cellular homeostasis, and altered metabolic pathway function is dependent upon both diet-sourced cofactors and genetic variation in the population. Further, SARS-CoV-2 infection is associated with low levels of NAD+ and strikingly dysregulates the NAD+ gene set for NAD+ synthesis and utilization, further emphasizing the importance of cofactor balancing for optimal health. Our hypothesis is that dysregulation of NADK (causing either increased or reduced oxidative stress) and its allied pathways may manifest as cancer or protection against cancer (healthy aging) in a cell-intrinsic manner, potentially dependent on genetic variation involving NADK and other genes in these linked pathways. To investigate this theory, we will assess the cellular biochemical impact of NAD+ supplementation in both healthy and PaCa cells by untargeted metabolomics, defining the cellular metabolomic impact of NAD+ supplementation and testing whether NAD+ depletion in combination with gemcitabine treatment enhances cancer cell death. We will also define human variation in NADK and genes that function in glutamine response, NAD biosynthesis, and pentose phosphate pathways, all important for NADPH generation. These data will improve our understanding of the broader cellular impact of NAD+ supplementation and depletion in both healthy and PaCa cells, as well as genetic variation in NAD+-associated pathways that may identify potential risk for metabolic dysfunction. This study is significant because it will investigate fully the NAD+ metabolome in a cell intrinsic manner and will define specific allelic differences in enzymes that regulate cofactor utilization and function in redox metabolic control/balance that may underlie an individual's risk for disease (e.g., cancer, infection) versus maintaining healthy aging.